A comparative evaluation of the turnaround time (TAT) of positive blood culture before and after matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) introduction in the laboratory routine was performed. A total of 643 positive blood cultures, of which 310 before and 333 after MALDI-TOF technique introduction, were collected. In the post MALDI-TOF period, blood culture median TAT decreased from 73.53 hours to 71.73 for Gram-positive, from 64.09 hours to 63.59 for Gram-negative and from 115.7 hours to 47.62 for anaerobes. MALDI-TOF significantly decreased the TAT of anaerobes, for which antimicrobial susceptibility test is not routinely performed. Furthermore, the major advantage of MALDI-TOF introduction was the decrease of the time for pathogen identification (TID) independently from the species with an improvement of 93% for Gram-positive, 86% for Gram-negative and 95% for anaerobes. In addition, high species-level identification rates and cost savings than conventional methods were achieved after MALDI-TOF introduction.